Abstract

The aim of the current study is to determine whether butein (3,4,2',4'-tetrahydroxychalcone) exhibits antiproliferative effects against tumor cells through suppression of the signal transducer and activator of transcription 3 (STAT3) activation pathway. We investigated the effects of butein on constitutive and inducible STAT3 activation, role of tyrosine kinases and phosphatases in STAT3 activation, STAT3-regulated gene products, and growth modulation of tumor cells. We found that this chalcone inhibited both constitutive and interleukin-6-inducible STAT3 activation in multiple myeloma (MM) cells. The suppression was mediated through the inhibition of activation of the upstream kinases c-Src, Janus-like kinase (JAK) 1, and JAK2. Vanadate treatment reversed the butein-induced down-regulation of STAT3 activation, suggesting the involvement of a tyrosine phosphatase. Indeed, we found that butein induced the expression of the tyrosine phosphatase SHP-1 and deletion of SHP-1 gene by small interfering RNA abolished the ability of butein to inhibit STAT3 activation, suggesting the critical role of SHP-1 in the action of this chalcone. Butein down-regulated the expression of STAT3-regulated gene products such as Bcl-xL, Bcl-2, cyclin D1, and Mcl-1, and this led to the suppression of proliferation and induction of apoptosis. Consistent with these results, overexpression of constitutive active STAT3 significantly reduced the butein-induced apoptosis. Moreover, we found that butein significantly potentiated the apoptotic effects of thalidomide and Velcade in MM cells. Overall, these results suggest that butein is a novel blocker of STAT3 activation and thus may have potential in suppression of tumor cell proliferation and reversal of chemoresistance in MM cells.

Butein inhibits constitutively active STAT3 in U266 cells. A, the chemical structure of butein (2′,4′,3,4-tetrahydroxychalcone). B, butein suppresses phospho-STAT3 levels in a dose-dependent manner. U266 cells (2 × 106/ml) were treated with the indicated concentrations of butein for 4 h, after which whole-cell extracts were prepared, and 30 μg of protein was resolved on 7.5% SDS-PAGE gel, electrotransferred onto nitrocellulose membranes, and probed for phospho-STAT3. C, butein suppresses phospho-STAT3 levels in a time-dependent manner. U266 cells (2 × 106/ml) were treated with 50 μM butein for the indicated durations and analyzed for phospho-STAT3 levels. The results shown are representative of three independent experiments. D, U266 cells (2 × 106/ml) were treated with the indicated concentrations of butein for 4 h and analyzed for nuclear STAT3 levels by EMSA. E, U266 cells (2 × 106/ml) were treated with 50 μM butein for the indicated durations and analyzed for nuclear STAT3 levels by EMSA. F, butein causes inhibition of translocation of STAT3 to the nucleus. U266 cells (1 × 105/ml) were incubated with or without 50 μM butein for 4 h and then analyzed for the intracellular distribution of STAT3 by immunocytochemistry. The same slides were counterstained for nuclei with Hoechst (50 ng/ml) for 5 min. G, SCC4 (2 × 106/ml) and DU145 (2 × 106/ml) cells were incubated with 50 μM butein for 4 h, and nuclear extracts were prepared and analyzed for STAT3 activation by EMSA. The results shown are representative of three independent experiments.

Butein down-regulates IL-6-induced phospho-STAT3 and Akt. A, MM.1S cells (2 × 106/ml) were treated with IL-6 (10 ng/ml) for the indicated time, whole-cell extracts were prepared, and phospho-STAT3 was detected by Western blot. The same blots were stripped and reprobed with STAT3 antibody to verify equal protein loading. B, MM.1S cells (2 × 106/ml) were treated with IL-6 with the indicated concentrations for 15 min, whole-cell extracts were prepared, and phospho-STAT3 was detected by Western blot. The same blots were stripped and reprobed with STAT3 antibody to verify equal protein loading. C, MM.1S cells (2 × 106/ml) were treated with 50 μM butein for the indicated times and then stimulated with IL-6 (10 ng/ml) for 15 min. Whole-cell extracts were then prepared and analyzed for phospho-STAT3 by Western blotting. The same blots were stripped and reprobed with STAT3 antibody to verify equal protein loading. The results shown are representative of three independent experiments. D, MM.1S cells (2 × 106/ml) were treated with 50 μM butein for the indicated times and then stimulated with IL-6 (10 ng/ml) for 15 min. Whole-cell extracts were then prepared and analyzed for phospho-Akt by Western blotting. The same blots were stripped and reprobed with Akt antibody to verify equal protein loading.

A, Butein suppresses phospho-Src levels. U266 cells (2 × 106/ml) were treated with the indicated concentrations of butein, after which whole-cell extracts were prepared, and 30-μg aliquots of those extracts were resolved on 10% SDS-PAGE, electrotransferred onto nitrocellulose membranes, and probed for phospho-Src antibody. The same blots were stripped and reprobed with Src antibody to verify equal protein loading. The blots were quantitated, and relative induction is mentioned as a -fold. B, butein suppresses phospho-JAK1 expression. U266 cells (2 × 106/ml) were treated with the indicated concentrations of butein, after which whole-cell extracts were prepared and 30-μg aliquots of those extracts were resolved on 10% SDS-PAGE, electrotransferred onto nitrocellulose membranes, and probed for phospho-JAK1 antibody. The same blots were stripped and reprobed with JAK1 antibody to verify equal protein loading. The blots were quantitated, and relative induction is mentioned as a -fold. C, butein suppresses JAK2 activity. U266 cells (2 × 106/ml) were treated with the indicated concentrations of butein, after which whole-cell extracts were prepared, and kinase assay was performed. The blots were quantitated, and relative induction is mentioned as a -fold. The results shown are representative of three independent experiments.

A and B, pervanadate reverses the phospho-STAT3 inhibitory effect of butein. A, U266 cells (2 × 106/ml) were treated with the indicated concentration of pervanadate and 50 μM butein for 4 h, after which whole-cell extracts were prepared, and 30-μg portions of those extracts were resolved on 7.5% SDS-PAGE gel, electrotransferred onto nitrocellulose membranes, and probed for phospho-STAT3 and STAT3. B, pervanadate affects the phosphorylation level of STAT3 in the presence or absence of butein. C and D, butein induces the levels SHP-1 and mRNA expression in U266 cells. Cells (2 × 106/ml) were treated with the indicated concentrations of butein for 4 h; after completion of incubation, either whole-cell extract or RNA was isolated. Portions (30 μg) of those extracts were resolved on 10% SDS-PAGE, electrotransferred onto nitrocellulose membranes, and probed for SHP-1 antibody. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading. RNA samples were subjected to RT-PCR with SHP-1 specific primers. E and F, effect of SHP-1 knockdown on butein-induced expression of SHP-1. SCC4 cells (1 × 105/ml) were transfected with either SHP-1-specific or scrambled siRNA (50 nM). After 48 h, cells were treated with 50 μM butein for 4 h, and whole-cell extracts were subjected to Western blot analysis for SHP-1. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading (E), and transfection with SHP-1 siRNA reverses butein-induced suppression of STAT3 activation. The same whole-cell extracts were subjected to phospho-STAT3 and STAT3 (F). G, the effect of butein on SH-PTP2. U266 cells (2 × 106/ml) were treated with the indicated concentrations of butein, after which whole-cell extracts were prepared, and Western blot was performed against SH-PTP2 antibody. The same blot was reprobed with β-actin antibody to verify equal protein loading. The results shown are representative of three independent experiments.

A, butein down-regulates the expression of antiapoptotic proteins. U266 cells (2 × 106/ml) were treated with 25 μM butein for the indicated time intervals, cells were harvested after incubation, and whole-cell extracts were prepared; 30-μg portions of those extracts were resolved on 10% SDS-PAGE and probed against Bcl-xL, Bcl-2, cyclin D1, and Mcl-1 antibodies. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading. The blots were quantitated, and relative induction is mentioned as a -fold. B, butein inhibits gene expression. U266 cells (106/ml) were treated with butein (25 μM) for the indicated times, and total RNA was extracted and examined for the expression of Bcl-2 and Bcl-xL by RT-PCR. GAPDH was used as an internal control to show equal RNA loading. C and D, cytotoxic effects of butein on U266 (C) and MM.1S (D) cells. Cells were plated in triplicate, treated with indicated concentrations of butein for 72 h, and then subjected to MTT assay to analyze the viability of cells. E and F, butein is more effective than AG490. U266 cells were plated in triplicate, treated with the indicated concentrations of butein and AG490 for 24 h, and then subjected to MTT assay (E). U266 cells were treated with 50 μM concentration of butein and 100 μM AG490 for 4 h, whole-cell extracts were prepared, and 30-μg portions of those extracts were resolved on 10% SDS-PAGE and probed against p-STAT3 and STAT3 (F). The results shown are representative of three independent experiments.

A, knockdown of STAT3 siRNA inhibited the apoptotic effect of butein. SCC4 cells were transfected with either STAT3-specific or scrambled siRNA (50 nM). After 48 h, cells were treated with 25 μM butein for 24 h and analyzed for the percentage of apoptosis by Live/Dead assay, and 20 random fields were counted. B and C, overexpression of constitutive STAT3 rescues A293 cells from butein-induced cytotoxicity. First, A293 cells were transfected with constitutive STAT3 plasmid. The cells were harvested 24 h after transfection, and the transfection was confirmed by Western blot analysis (B). A293 cells were transfected with constitutive STAT3 plasmid. After 24 h of transfection, the cells were treated with 25 μM butein for 24 h, and then the cytotoxicity was determined by Live/Dead assay, and 20 random fields were counted (C). D, butein potentiates the apoptotic effect of thalidomide and Velcade. U266 cells (1 × 106/ml) were treated with 25 μM butein and 10 ng/ml thalidomide or 20 nM Velcade alone or in combination for 24 h at 37 °C. Cells were stained with a Live/Dead assay reagent for 30 min and then analyzed under a fluorescence microscope, and 20 random fields were counted. The results shown are representative of three independent experiments.